Volume 127, Issue 3, Pages 924-936 (September 2004) High resolution analysis of cellular immune responses in resolved and persistent hepatitis C virus infection  Georg M. Lauer, Eleanor Barnes, Michaela Lucas, Joerg Timm, Kei Ouchi, Arthur Y. Kim, Cheryl L. Day, Gregory K. Robbins, Deborah R. Casson, Markus Reiser, Geoffrey Dusheiko, Todd M. Allen, Raymond T. Chung, Bruce D. Walker, Paul Klenerman  Gastroenterology  Volume 127, Issue 3, Pages 924-936 (September 2004) DOI: 10.1053/j.gastro.2004.06.015 Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 1 Example of screening for HCV-specific CD8+ T-cell responses. The strength and specificity of the HCV specific CD8+ T-cell response was determined in each individual using an interferon-γ ELISpot matrix spanning the entire HCV genome. Responses in a representative patient (R5) are shown here. The epitopes targeted are plotted beneath the corresponding position in the HCV genome map, and the magnitude of the response (SFC, spot-forming cells) is given. Subject R5 targeted 6 different epitopes, 1 located in E1, 1 in NS3, 2 in NS4, and 2 in NS5 (A). Strong responses, such as the one directed against the peptide NS4-1745, could be confirmed in a direct ex vivo ICS (B). Responses were confirmed as CD8 positive using peptide-specific T-cell lines (following a single round of peptide stimulation; C), and HLA restriction of the epitope was defined using partially HLA-matched and -mismatched heterologous B cell lines (C). Gastroenterology 2004 127, 924-936DOI: (10.1053/j.gastro.2004.06.015) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 2 Comparison of the strength and breadth of the HCV-specific CD8+ T-cell response. In each individual, the summation of spot-forming cells (SFC) for all epitopes targeted by that individual (A) and the number of epitopes targeted by each individual (B) are plotted, and the 2 cohorts are compared using the Mann-Whitney nonparametric test. Open squares and closed triangles represent individuals with chronic and resolved infection, respectively. Gastroenterology 2004 127, 924-936DOI: (10.1053/j.gastro.2004.06.015) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 3 Distribution of HCV epitopes and frequency of recognition. Figure 3A shows the distribution of HCV-specific responses over the HCV genome in the entire cohort (n = 40). Each bar represents a distinctive HCV epitope, with the length of the bar indicating the number of subjects targeting this epitope. Bars above the line correspond to responses in the cohort with resolved infection, and those below the line represent responses detected in persons with chronic infection. Figure 3B shows recognition of specific epitopes known to be restricted by HLA alleles expressed by at least 3 persons in each cohort. Epitopes included were selected based on recognition in this study or because they had been described as being frequently recognized in previous studies. The bars indicate the percentage of subjects bearing the respective HLA allele who recognized the epitope in the ELISpot assay. Gastroenterology 2004 127, 924-936DOI: (10.1053/j.gastro.2004.06.015) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 4 Ex vivo HCV tetramer response. Tetramers were custom synthesized for 7 different HCV epitopes and restricted by 4 different HLA alleles, and cells were stained ex vivo in both resolved and unresolved infection. The percentage of tetramer+ cells/total CD8+ cells is given in the upper right panel of each dot plot. Gastroenterology 2004 127, 924-936DOI: (10.1053/j.gastro.2004.06.015) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 5 Phenotype of HCV-specific CD8+ T cells. (A) Analysis of phenotypic marker expression across all tetramer responses analyzable. Triangles represent individuals with resolved infection, and squares represent individuals with chronic infection. For the chronic subjects, responses marked by solid squares represent those reactive with the autologous infecting virus of the individual. Open squares mark responses not recognizing the circulating autologous virus. (B) The surface expression pattern of HCV-specific CD8+ T cells was analyzed using antibodies for CD45RA, CD27, CD28, and CCR7 and is shown for 3 epitopes in 2 representative patients (C2 and R3). The panels on the left show the expression of CD27 and CD28 within the total CD8+ T-cell population and the tetramer-positive cells alone. The panels on the right show CD45RA and CCR7 expression in the tetramer-positive and -negative populations. The 2 upper rows are representative examples for the phenotype seen in both chronic and resolved subjects. In contrast, the lower row shows the staining for a unique response in a single patient with a distinct phenotype. Note that the middle and lower rows are different epitopes recognized by the same individual with resolved infection. Gastroenterology 2004 127, 924-936DOI: (10.1053/j.gastro.2004.06.015) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 6 Correlation between tetramer-positive T cells and IFN-γ secretion. The number of tetramer-positive cells was correlated with the number of IFN-γ producing specific cells as detected in the ELISpot assay ex vivo (SFC, spot forming cells). The number of IFN-γ-secreting cells was universally lower, representing 10%–20% of tetramer-positive cells in individuals with both resolved and chronic infection. Gastroenterology 2004 127, 924-936DOI: (10.1053/j.gastro.2004.06.015) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 7 HCV-specific CD8+ T cells can proliferate and lyse target cells after a single round of peptide stimulation in vitro in individuals with both resolved and chronic infection. HCV-specific CD8+ T cells from subjects with resolved, as well as chronic infection could be expanded after 10–14 days, following a single round of peptide stimulation (A) in individuals with both resolved and chronic infection. Peptide specificity was confirmed by ICS for IFN-γ The cell lines specifically lysed peptide pulsed targets in a standard 4-hour chromium release assay at an effector/target ratio 30:1 (B). Gastroenterology 2004 127, 924-936DOI: (10.1053/j.gastro.2004.06.015) Copyright © 2004 American Gastroenterological Association Terms and Conditions